The overall goal of this research is to understand the regulation of growth hormone (GH) and prolactin (Prl) gene expression in developmental, cellular and molecular terms. We wish to define the sequences and identify the factors that mediate steroid and polypeptide hormonal regulation of gene expression. In vitro mutagenesis coupled with expression assays will be used to define regulatory regions. An exonuclease scan procedure will be used to locate regulatory proteins on DNA. Sequences conferring tissue specific expression will be mapped by transfection into differentiated endocrine cell lines. We will continue our analysis of sequences in GH fusion genes that promote cell specific expression in the brains of transgenic mice. A novel approach utilizing the fluorescence activated cell sorter (FACS) will be used to isolate transfected genomic DNA encoding regulatory proteins. To begin to understand, at a molecular level, how DNA binding proteins might regulate the expression of specific genes, we propose to clone, by expression, the human glucocorticoid receptor gene. This gene will be transduced via retroviral vectors into appropriate cell types in a manner that will allow selection in vivo of interesting mutations defining regulatory domains of the receptor. Characterization of these mutants will contribute to a definition of the mechanisms by which the glucocorticoid receptor influences gene expression.